Vγ4 γδ T Cell-Derived IL-17A Negatively Regulates NKT Cell Function in Con A-Induced Fulminant Hepatitis

Na Zhao, Jianlei Hao, Yuanyuan Ni, Wei Luo, Ruifang Liang, Guangchao Cao, Yapu Zhao, Puyue Wang, Liqing Zhao, Zhigang Tian, Richard Flavell, Zhangyong Hong, Jihong Han, Zhi Yao, Zhenzhou Wu and Zhinan Yin
J Immunol November 15, 2011, 187 (10) 5007-5014; DOI: https://doi.org/10.4049/jimmunol.1101315

Abstract

Con A-induced fulminant hepatitis is a well-known animal model for acute liver failure. However, the role of γδ T cells in this model is undefined. In this report, using TCR δ−/− mice, we demonstrated a protective role of γδ T cells in Con A-induced hepatitis model. TCR δ−/− mice showed significantly decreased levels of IL-17A and IL-17F in the Con A-treated liver tissue, and reconstitution of TCR δ−/− mice with wild-type (Wt), but not IL-17A−/−, γδ T cells significantly reduced hepatitis, strongly suggesting a critical role of IL-17A in mediating the protective effect of γδ T cells. Interestingly, only Vγ4, but not Vγ1, γδ T cells exerted such a protective effect. Furthermore, depletion of NKT cells in TCR δ−/− mice completely abolished hepatitis, and NKT cells from Con A-challenged liver tissues of TCR δ−/− mice expressed significantly higher amounts of proinflammatory cytokine IFN-γ than those from Wt mice, indicating that γδ T cells protected hepatitis through targeting NKT cells. Finally, abnormal capacity of IFN-γ production by NKT cells of TCR δ−/− mice could only be downregulated by transferring Wt, but not IL-17−/−, Vγ4 γδ T cells, confirming an essential role of Vγ4-derived IL-17A in regulating the function of NKT cells. In summary, our report thus demonstrated a novel function of Vγ4 γδ T cells in mediating a protective effect against Con A-induced fulminant hepatitis through negatively regulating function of NKT cells in an IL-17A–dependent manner, and transferring Vγ4 γδ T cells may provide a novel therapeutic approach for this devastating liver disease.

Con A-induced hepatitis is a well-documented murine model that closely resembles the pathology of human hepatitis (1). It has been demonstrated that NKT cells play an essential role in the pathogenesis of Con A-induced liver injury through secreting IFN-γ and other inflammatory cytokines (2). Indeed, either deletion of NKT cells using CD1d−/− or Vα14−/− mice, or blocking IFN-γ and its signaling pathway with IFN-γ−/− or stat1−/− mice completely abolishes Con A-induced liver damage (2–7). However, the mechanisms that control cytokine production by NKT cells are largely unknown.

γδ T cells have many unique features and functions (8–10). γδ T cells can recognize both self- and nonself-ligands (11–13); therefore, they are divided into two subsets: “Ag-experienced” γδ T cells are prone to produce IFN-γ, whereas “Ag-naive” γδ T cells produce IL-17 as the innate source of this cytokine (14). Consistently, our previous studies have demonstrated that splenic γδ T cells predominantly produce IFN-γ on activation (15–17) and play an important role in tumor immunity (18, 19). According to the TCR usage, peripheral γδ T cells are composed of two main subsets: Vγ1 γδ T cells and Vγ4 γδ T cells (20–22). These two subsets have divergent functions in many disease models (23–27). Our recent work has defined a protective role of Vγ4 γδ T cells in tumor immunity through Eomes-controlled IFN-γ production (19). Recently, it has been reported from several models that Vγ4 γδ T cells are the main source of IL-17 in periphery (27, 28). However, the role of this subset of γδ T cells in Con A-induced NKT cell-mediated hepatitis is completely unknown.

In this report, we demonstrated for the first time, to our knowledge, that γδ T cells, especially Vγ4 γδ T cells, played a protective role in Con A-induced hepatitis model through providing IL-17A, which, in turn, negatively regulates the function of NKT cells, especially their IFN-γ production.

Materials and Methods

Mice

All experiments were performed with age- (6–8 wk) and sex-matched mice (male or female mice were used). C57BL/6J (B6 wild-type [Wt]) mice were purchased from Academy of Military Medical Science (Beijing, China). B6.129P2-Tcrdtm1Mom (B6 TCR δ−/−) mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and C57BL/6 IL-17A−/− mice (29) were kindly provided by Dr. Richard Flavell.

Reagents

Con A (type IV) was purchased from Sigma Chemical (St. Louis, MO). Recombinant mouse (rm) IL-2 was purchased from R&D Systems (Minneapolis, MN), and rmIL-17 was from eBioscience (San Diego, CA). FITC-conjugated anti-mouse TCR β, allophycocyanin-conjugated anti-mouse CD4 (clone GK1.5), anti-mouse NK1.1 (clone PK136), purified anti-mouse IL-17A (clone 17F3), hamster anti-mouse TCR Vγ1 mAb 2.11, and hamster anti-mouse TCR Vγ4 mAb UC3 were from Sungene (Tianjin, China); Alexa Fluor 647-conjugated anti-mouse γδ TCR (clone UC7) and allophycocyanin-conjugated anti-mouse IFN-γ (clone XMG1.2) were purchased from BioLegend (San Diego, CA). PE-conjugated anti-mouse IL-17A (clone TC11-18H10) was purchased from BD Biosciences (San Jose, CA).

T cell-mediated hepatitis model and survival study

To induce hepatitis, we injected Wt, TCR δ−/−, and IL-17A−/− mice i.v. with Con A (10 mg/kg body weight). For survival study, the dose of Con A was increased to 20 mg/kg body weight. Mice were closely monitored and euthanized before the end point was reached.

Assay for serum transaminase activity

Mice serum samples were obtained at different time points after Con A injection. Serum alanine aminotransferase (ALT) activities were measured using commercial available test kit (Rong Sheng Biotech, Shanghai, China) based on methods recommended by the International Federation of Clinical Chemistry.

Histology

Liver tissues were harvested after treatment of Con A, fixed in 10% formalin, and embedded in paraffin. Five-micrometer sections were affixed to slides, stained with H&E, and images were acquired on a Leica DM3000 microscope using 10× objective. The liver damage extent was quantified by the necrosis area as a percentage of total area using Image-Pro Plus (IPP) 6.0 software (Media Cybernetics, Silver Spring, MD). Three different fields were randomly chosen in every slide, and average necrosis percentage was calculated.

Liver mononuclear cell preparation

Mouse livers were removed and pressed through a 200-gauge stainless-steel mesh. The liver cell suspension was collected, suspended in PBS, and centrifuged at 50 × g for 5 min. Supernatants containing mononuclear cells (MNCs) were collected and resuspended in 40% Percoll (GE Healthcare). The cell suspension was gently overlaid onto 70% Percoll and centrifuged for 30 min at 1260 × g. MNCs were collected from the interphase and washed twice in PBS.

Adoptive transfer experiments

For expansion of γδ, Vγ1, and Vγ4 γδ T cells in vitro, γδ T cells were sorted and expanded from splenocytes either from Wt or IL-17A−/− mice as described previously (19). Expanded cells (1 × 106 cells/mouse) were i.v. transferred into B6 TCR δ−/− mice 24 h before Con A administration.

Intracellular cellular cytokine staining

For IL-17A staining, liver lymphocytes were isolated 2 h after Con A treatment and stimulated with PMA (50 ng/ml; Sigma, St. Louis, MO) and ionomycin (1 μg/ml; Sigma) in the presence of GolgiPlug (BD Biosciences, San Jose, CA) for 4.5 h. Cells were stained first with Abs against surface molecules and then fixed and permeabilized as described previously (15). For IFN-γ staining, liver lymphocytes from different groups of mice were isolated 12 h after Con A treatment, and stimulated with plate-coated anti-CD3 and soluble anti-CD28 and IL-2 for 6 h in the presence of GolgiPlug. Cells were then stained with FITC–anti-mouse TCR β and PE–anti-mouse NK1.1 followed by fixation, permeabilization, and intracellular staining as described previously (15).

Cell depletion

Mice were administered with an i.v. injection of 50 μl anti-asialo GM1 (ASGM1; Wako Pure Chemical Industries, Osaka, Japan) diluted in 200 μl pyrogen-free PBS to deplete NK cells. To deplete both NK and NKT cells, we injected mice i.v. with 200 μg anti-NK1.1 mAb (PK136; American Type Culture Collection) diluted in 200 μl pyrogen-free PBS. Depletion was confirmed by flow cytometry. For neutralizing effect of IL-17A in vivo, anti-mouse IL-17A Ab (100 μg/mouse) was injected i.v. 1 h before Con A treatment. Mice with cell depletion were then treated with Con A as described earlier.

Real-time PCR for gene transcription

Total RNA was extracted from liver MNCs by using TRIzol Reagent (Invitrogen, Carlsbad, CA) and reverse-transcribed by Quantscript RT Kit (Tiangen, Beijing, China). mRNA expression was quantified by SYBR Premix Hotmaster Taq (Tiangen, Beijing, China), and ribosomal protein large P0 (RPLP0) (30) was used as an internal normalizing gene. The primer sequences used were as follows: IL-17A forward: 5′-TGA AGG CAG CAG CGA TCA-3′, reverse: 5′-GGA AGT CCT TGG CCT CAG TGT-3′; IL-17F forward: 5′-CGC CAT TCA GCA AGA AAT CC-3′, reverse: 5′-CTC CAA CCT GAA GGA ATT AGA ACA G-3′; IFN-γ forward: 5′-ATG AAC GCT ACA CAC TGC ATC-3′, reverse: 5′-CCA TCC TTT TGC CAG TTC CTC-3′; IL-4 forward: 5′-GAA AAC TCC ATG CTT GAA GAA-3′, reverse: 5′-TCT TTC AGT GAT GTG GAC TTG 3′; TNF-α forward: 5′-CTA CTG AAC TTC GGG GTG AT-3′, reverse: 5′-CAG GCT TGT CAC TCG AAT T-3′; T-bet forward: 5′-TTT CAT TTG GGA AGC TAA AG-3′, reverse: 5′-GGC TGG TAC TTG TGG AGA GA-3′; GATA-3 forward: 5′-AGA GGT GGA CGT ACT TTT TAA C-3′, reverse: 5′-AGA GAT CCG TGC AGC AGA G-3′; FASL forward: 5′-CAG CTT CAG ATG CAA GTG AGT GG-3′, reverse: 5′-CAA GGA CAG AAC TCT GAC GCT GAC-3′; RPLP0 forward: 5′-GAA ACT GCT GCC TCA CAT CCG-3′, reverse: 5′-CTG GCA CAG TGA CCT CAC ACG-3′.

Statistics

Data are presented as mean values ± SD. Statistical significance between two groups was evaluated by two-tailed unpaired Student t test using InStat version 3.06 software for Windows (GraphPad, San Diego, CA). For comparing values obtained in three or more groups, we used one-factor ANOVA, followed by S-N-K’s post hoc test. The difference of survival was compared and analyzed using the log-rank test, performed by GraphPad Prism 4 for Windows (GraphPad). Throughout the text, figures, and figure legends, the following terminology is used to denote statistical significance: *p < 0.05, **p < 0.01.

Results

γδ T cells play a protective role in Con A-induced fulminant hepatitis

To define the role of γδ T cells in Con A-induced hepatitis, sex- and age-matched B6 Wt and TCR δ−/− mice were treated with high dose of Con A (20 mg/kg body weight), and the survival rate of mice was observed and recorded. In comparison with B6 Wt mice, TCR δ−/− mice died much earlier and no mice survived after 20 h (Fig. 1A), indicating an essential role of γδ T cells in protection against Con A-induced liver damage. To further dissect the protective role of γδ T cells in this model, we administered different doses of Con A to B6 Wt or TCR δ−/− mice, and a lower dose of Con A (10 mg/kg body weight) was found to be the best to demonstrate significant differences between Wt and TCR δ−/− mice (data not shown). Thereafter, all the following experiments were performed using this lower dose. On treatment with Con A, hepatitis appeared significantly earlier and lasted much longer in TCR δ−/− mice in comparison with Wt mice, as indicated by the serum ALT level (Fig. 1B). This was further evidenced by liver histopathology at 18 h after treatment of Con A (Fig. 1C, 1D). Significantly bigger areas of necrosis in liver tissues were observed in TCR δ−/− mice.

FIGURE 1.
FIGURE 1.

γδ T cells play a protective role in Con A-induced hepatitis. A, Sex- and age-matched B6 Wt and TCR δ−/− mice (n = 15 for each group) were i.v. injected with Con A (20 mg/kg body weight), and the rates of death were observed every 30 min and recorded. One representative experiment of three repeated experiments is shown (*p < 0.05). B, Sex- and age-matched B6 Wt and TCR δ−/− mice (n = 12 for each group) were i.v. injected with Con A (10 mg/kg body weight), and at different time points after Con A treatment, serum samples were obtained for measuring the level of ALT. One representative experiment is shown (**p < 0.01). C, Liver tissues at 18 h after Con A treatment were fixed for H&E staining, and one representative tissue staining is shown. N, necrosis area. Scale bars, 200 μm. D, Percentage of necrosis is calculated and shown. n = 3 mice/group. **p < 0.01. E, B6 TCR δ−/− mice (n = 6) were reconstituted with in vitro-activated total γδ T cells (1 × 106 cells/mouse) as described in Materials and Methods, followed with Con A treatment. Serum samples were obtained at different time points after Con A treatment, and the levels of ALT were measured. One of three repeated experiments is shown (*p < 0.05, **p < 0.01).

To further define the role of γδ T cells in Con A-induced hepatitis, we adopted an another approach. B6 TCR δ−/− mice were reconstituted with in vitro-activated Wt γδ T cells as described in Materials and Methods. Adoptive transfer of γδ T cells into TCR δ−/− mice significantly reduced hepatitis (Fig. 1E), further confirming the protective role of γδ T cells. These results collectively demonstrated a protective role of γδ T cells in Con A-induced hepatitis model.

IL-17A is a critical cytokine for protection against Con A-induced hepatitis

IL-17 cytokine family has multiple members and it has been shown to be involved in many inflammatory diseases. To define the role of IL-17A in Con A-induced hepatitis, we treated sex- and age-matched B6 Wt and IL-17A−/− mice with Con A and measured serum level of ALT at different time points. In the absence of IL-17A, significantly worse inflammation was observed on Con A treatment, as indicated by the significantly increased level of ALT (Fig. 2A) and bigger necrosis areas shown by liver histopathology (Fig. 2B, 2C), suggesting a protective role of IL-17A in this hepatitis model. B6 IL-17A−/− mice were administered with rmIL-17 and serum ALT level was monitored to further confirm the protective effect of IL-17A. Reconstitution of IL-17A−/− mice with IL-17 resulted in protection against Con A-induced hepatitis, evidenced by serum ALT level (Fig. 2D) and pathology (Fig. 2E, 2F), supporting the protective effect of IL-17 in this fulminant hepatitis model. Consistently, treatment of B6 Wt mice with an anti–IL-17A Ab rendered these mice more susceptible to Con A treatment (Fig. 2G). Therefore, our results collectively demonstrate a protective role of IL-17A in this hepatitis model.

FIGURE 2.
FIGURE 2.

IL-17A is required for protection against Con A-induced liver damage. A, Sex- and age-matched B6 Wt and IL-17A−/− mice (n = 15 for each group) were i.v. injected with Con A (10 mg/kg body weight), and at different time points after Con A treatment, serum samples were obtained for measuring the level of ALT. One of three repeated experiments is shown (**p < 0.01). B, Liver tissues of B6 Wt or IL-17A−/− mice at 18 h after Con A treatment were fixed and stained with H&E. One representative staining for each strain of mice is shown. Scale bars, 200 μm. C, Percentage of necrosis is shown. n = 3 mice/group. **p < 0.01. D, B6 IL-17A−/− mice (n = 5) were treated with i.p. injection of rmIL-17 (1 μg/mouse) or PBS vehicle 30 min before and 1 h after Con A treatment, and serum ALT levels were measured from serum samples 18 h after Con A treatment. One representative experiment is shown (**p < 0.01). E, Liver tissues of B6 IL-17A−/− mice that received PBS or rmIL-17 at 18 h after Con A administration were fixed and stained with H&E. One representative staining is shown. Scale bars, 200 μm. F, Percentage of necrosis is shown. n = 3 mice/group. **p < 0.01. G, For IL-17A blocking, B6 Wt mice (n = 5) were treated with i.v. injection of either anti-mouse IL-17A Ab, 100 μg/mouse, or control Ab 1 h before Con A treatment. Serum samples were collected at different time points after Con A administration for analysis of the ALT level. One representative experiment is shown (*p < 0.05, **p < 0.01). N, necrosis area.

γδ T cells are the primary source of IL-17A on Con A treatment

Based on our findings as described earlier that both γδ T cells and IL-17A played important roles in protection against Con A-induced hepatitis, we hypothesized that γδ T cells might be the primary source of IL-17A. To test our hypothesis, we treated sex- and age-matched B6 Wt or TCR δ−/− mice with Con A (10 mg/kg), and at 2 h after treatment, liver lymphocytes were collected for RNA extraction and an array of cytokines were analyzed by real-time PCR. In comparison with Wt mice, liver lymphocytes of TCR δ−/− mice expressed significantly lower level of IL-17A and IL-17F (Fig. 3A). To further confirm the primary source of IL-17A, we isolated liver lymphocytes from Wt mice at 2 h after Con A treatment and activated them, followed by intracellular staining as described in Materials and Methods. Interestingly, >30% of liver γδ T cells expressed IL-17A, and few CD4+ T and TCR αβ+ T cells showed IL-17A+ (Fig. 3B), supporting the notion that γδ T cells, but not Th17 or other αβ+ T cells (such as NKT cells), were the predominant source of IL-17A in Con A-induced hepatitis.

FIGURE 3.
FIGURE 3.

γδ T cells are the primary source of IL-17A in Con A-induced hepatitis. Sex- and age-matched B6 Wt and TCR δ−/− mice were treated with Con A (10 mg/kg body weight) as described earlier, and liver lymphocytes were isolated at 2 h after Con A treatment for gene expression analyzed by real-time PCR (A, C) (n = 5 for each group; *p < 0.05, **p < 0.01) and intracellular cytokine staining (B) (pooled cells). Data are representative of three independent experiments.

In contrast, no significant changes were observed between these two groups of mice for IFN-γ, IL-4, TNF-α, T-bet, GATA-3, and FASL (Fig. 3C), suggesting a critical role of γδ T cells in providing the primary source of IL-17A in Con A-induced hepatitis model.

γδ T cell-derived IL-17A is critical for protective immune response against Con A-induced hepatitis

To test the hypothesis that the protective effect of γδ T cells is through providing IL-17A, we administered B6 TCR δ−/− mice with rmIL-17 as described earlier and monitored serum ALT level and liver histopathology. Indeed, injection of IL-17A significantly reversed Con A-induced hepatitis in TCR δ−/− mice (Fig. 4A), supporting a critical role of IL-17A in γδ T cell-mediated protective responses on Con A challenge.

FIGURE 4.
FIGURE 4.

γδ T cell-derived IL-17A is critical for protective immune response against Con A-induced hepatitis. A, B6 TCR δ−/− mice (n = 8) were treated with i.p. injection of either rmIL-17, 2 μg/mouse, or PBS vehicle 30 min before and 1 h after Con A treatment. Serum samples at 18 h after Con A treatment were used for analysis of the ALT level. One representative experiment is shown (**p < 0.01). B, B6 TCR δ−/− mice were reconstituted with cultured γδ T cells (1 × 106 cells/mouse) either from Wt or IL-17A−/− mice or PBS (n = 6 for each group) followed with the treatment of Con A as described earlier. At different time points after Con A treatment, serum samples were collected for determination of ALT level. One of three independent experiments is shown (*p < 0.05, **p < 0.01).

To further test the critical role of IL-17A derived from γδ T cells, we adopted a similar approach as described in Fig. 1E. Splenic γδ T cells were isolated either from Wt or IL-17A−/− mice, activated in vitro, and then transferred into TCR δ−/− mice (1 × 106 cells/mouse). At 24 h after transfer, these recipients were then treated with low dose of Con A as described earlier. Serum samples were collected at different time points for monitoring the ALT level, and liver tissues were collected at 18 h of Con A treatment for histopathology staining. Interestingly, TCR δ−/− mice reconstituted with Wt γδ T cells showed significant protection. In sharp contrast, reconstitution of TCR δ−/− mice with IL-17A−/− γδ T cells did not offer any protection (Fig. 4B); the severity of liver damage in these reconstituted mice was almost the same as untreated TCR δ−/− mice, strongly supporting a critical role of γδ T cell-derived IL-17A in mediating the protective effect of γδ T cells in Con A-induced hepatitis model.

Only Vγ4, but not Vγ1, γδ T cells offer protection against Con A-induced hepatitis

Peripheral γδ T cells are mainly composed of two subsets: Vγ1 and Vγ4. These two subsets of γδ T cells have divergent functions in many different disease models (9). Based on the results obtained earlier that IL-17A was a critical mediator for the protective effect of γδ T cells, and studies by others (27, 28, 31) and our own unpublished work that Vγ4 γδ T cells are the main producer of IL-17, we hypothesized that Vγ4 γδ T cells might be the critical one to play the protective effect. Vγ1 and Vγ4 γδ T cells were sorted from B6 Wt mice, cultured as described earlier, and used for IL-17 family cytokine analysis by real-time PCR to test this hypothesis. Only Vγ4, but not Vγ1, γδ T cells expressed high levels of IL-17A and IL-17F (Fig. 5A). Consistently, we found the main source of IL-17A in Con A-treated liver was the Vγ4+ subset (Fig. 5B). To directly compare the protective effect of Vγ1 and Vγ4 γδ T cells in vivo, we reconstituted TCR δ−/− mice with either Vγ1 or Vγ4 γδ T cells isolated from B6 Wt mice followed by Con A administration as described earlier. Only Vγ4 γδ T cells offered significant protection against Con A-induced hepatitis. In contrast, reconstitution with Vγ1 γδ T cells did not show any protective effect (Fig. 5C). To test whether the protective effect of Vγ4 γδ T cells was dose dependent, we transferred different amounts of Vγ4 γδ T cells to TCR δ−/− mice followed by Con A treatment. We showed that at least 1 million Vγ4 γδ T cells were required for the protective effect (Fig. 5D). The differential effects of these two subsets of γδ T cells were further confirmed by liver histopathology sections (Fig. 5E, 5F). To further prove the critical role of IL-17A in mediating the protective effect of Vγ4 γδ T cells, we reconstituted TCR δ−/− mice with either Wt or IL-17A−/− Vγ4 γδ T cells as described earlier, and monitored serum ALT level and liver histopathology. Consistently, only Wt, but not IL-17A−/−, Vγ4 γδ T cells showed significant protection. Reconstitution of Vγ4 γδ T cells significantly reduced serum ALT level (Fig. 5G). Therefore, our results determined a critical role of Vγ4 γδ T cells in protection against Con A-induced hepatitis through IL-17A production.

FIGURE 5.
FIGURE 5.

Only Vγ4, but not Vγ1, γδ T cells offer protection against Con A-induced hepatitis. A, Vγ1 and Vγ4 γδ T cells were sorted from B6 Wt mice, cultured as described earlier, and used for RNA extraction and gene expression analysis of real-time PCR. Data are representative of three independent experiments. B, B6 Wt mice were treated with Con A (10 mg/kg body weight), and lymphocytes were isolated from pooled liver tissues at 2 h after Con A treatment for intracellular IL-17A staining. Cells were gated on γδ TCR+ cells. C, B6 TCR δ−/− mice were reconstituted with either cultured Vγ1 or Vγ4 γδ T cells (1 × 106 cells/mouse) from B6 Wt mice, followed by Con A treatment as described earlier. The levels of ALT from serum samples at 18 h after treatment were measured. One representative experiment is shown (**p < 0.01). D, B6 TCR δ−/− mice were reconstituted with different number of cultured Vγ4 γδ T cells (n = 3 for each group) followed with the treatment of Con A as described previously. At 18 h after Con A treatment, serum samples were collected for determination of ALT level. One representative experiment is shown (**p < 0.01). E, Liver tissues of Con A-treated B6 TCR δ−/− mice at 18 h after Vγ1 or Vγ4 γδ T cell (1 × 106 cells/mouse) transfer as described previously were fixed and stained with H&E. One representative staining for each group of mice is shown. N, necrosis area. Scale bars, 200 μm. F, Percentage of necrosis is also shown (n = 3 mice/group, **p < 0.01). G, B6 TCR δ−/− mice were reconstituted with cultured Vγ4 γδ T cells from either Wt or IL-17A−/− mice, or with PBS, and then followed by Con A administration. The levels of ALT from serum samples at 18 h after treatment were measured. One representative experiment is shown (**p < 0.01).

Vγ4 γδ T cell-derived IL-17A regulates IFN-γ secretion by NKT cells

NKT cells play an essential role in the pathogenesis of Con A-induced hepatitis through producing inflammatory cytokines, especially IFN-γ (2). We hypothesized that the protective effect of γδ T cells might be through targeting NKT cells. To test our hypothesis, we treated TCR δ−/− mice with an anti-NK1.1 (clone PK136) or ASGM1 Ab to deplete NK1.1+ (include NK and NKT cells) or NK cells, respectively (32). These mice were then treated with Con A and the serum ALT level was measured at 18 h after treatment. Depletion of NK1.1+, but not NK, cells in TCR δ−/− mice significantly decreased Con A-induced liver damage, indicating an essential role of NKT cells in the Con A-induced hepatitis model, even in the absence of γδ T cells (Fig. 6A). Based on the fact that NKT cell-derived IFN-γ is critical for the pathogenesis of Con A-induced liver damage, we hypothesized that the protective effect of γδ T cell-derived IL-17A might negatively regulate IFN-γ production by NKT cells. To test our hypothesis, we treated sex- and age-matched B6 Wt and TCR δ−/− mice with Con A as described earlier. NKT and NK cells were sorted from liver at 2 h after Con A treatment, and the cytokine profiles of these NKT cells and NK cells were analyzed by real-time PCR. In comparison with those from Wt mice, NKT cells isolated from TCR δ−/− mice produced significantly higher levels of IFN-γ (Fig. 6B). In sharp contrast, no significant differences were observed in NK cell populations between these two mice, strongly supporting our hypothesis that γδ T cells indeed targeted NKT cells.

FIGURE 6.
FIGURE 6.

Vγ4 γδ T cell-derived IL-17 regulates IFN-γ secretion by NKT cells. A, B6 TCR δ−/− mice were treated with either anti-NK1.1 (clone PK136) or ASGM1 or control Ab 24 h before Con A treatment, and the level of ALT from serum samples obtained 18 h after Con A treatment was analyzed. One of three repeated experiments is shown (**p < 0.01). B, Total liver lymphocytes were isolated from Wt and TCR δ−/− mice 2 h after PBS or Con A treatment. NK and NKT cells sorted by flow cytometry were used for RNA extraction and analysis of IFN-γ expression level by real-time PCR. One representative experiment is shown. C, Sex- and age-matched B6 TCR δ−/− mice were divided into four groups, reconstituted with Wt Vγ4 γδ T cells (1 × 106 cells/mouse), IL-17A−/− Vγ4 γδ T cells (1 × 106 cells/mouse), PBS vehicle, and Wt Vγ4 γδ T cells (1 × 106 cells/mouse) plus anti-mouse IL-17A Ab (100 μg/mouse), respectively. These mice were then treated with Con A as described earlier. NKT cells were sorted from isolated total liver lymphocytes 2 h after Con A treatment for RNA extraction and analysis of the expression level of IFN-γ by real-time PCR. One repeatable experiment is shown. D, B6 TCR δ−/− mice were reconstituted with cultured Vγ4 γδ T cells from either Wt or IL-17A−/− mice, or with PBS, and then followed by Con A administration. B6 Wt mice were also used as control. Liver lymphocytes were isolated at 12 h after Con A treatment for intracellular cytokine staining. NKT cells (gated on TCR β+ NK1.1+) were analyzed for IFN-γ production. Results represent one of three repeated experiments.

To further define whether this regulatory effect of γδ T cells on NKT cells was IL-17A dependent, we reconstituted sex- and age-matched B6 TCR δ−/− mice with either Wt or IL-17A−/− Vγ4 γδ T cells, or left them untreated. These mice were then treated with Con A as described earlier. NKT cells were sorted from liver at 2 h after Con A treatment, and the cytokine profiles of these NKT cells were then analyzed by real-time PCR. Interestingly, NKT cells from TCR δ−/− mice reconstituted with Wt Vγ4 γδ T cells expressed a significantly lower level of IFN-γ, indicating a protective role of Vγ4 γδ T cells through altering the proinflammatory cytokine production by NKT cells (Fig. 6C). In contrast, NKT cells from mice reconstituted with IL-17A−/− Vγ4 γδ T cells showed a similar level of IFN-γ as those in TCR δ−/− mice (Fig. 6C). To further confirm the regulatory effect of Vγ4 γδ T cells on the ability of NKT cells to produce IFN-γ, we sorted NKT cells at 12 h after Con A treatment and activated them for intracellular cytokine staining. In our preliminary studies, at this time point, the biggest differences were observed between Wt and TCR δ−/− mice in their IFN-γ production by NKT cells (data not shown). In the absence of γδ T cells, NKT cells produce a significantly higher level of IFN-γ, which was inhibited by transferring Wt, but not IL-17A−/−, Vγ4 γδ T cells (Fig. 6D), suggesting a critical role of IL-17A in mediating the protective effect of Vγ4 γδ T cells through targeting IFN-γ production by NKT cells.

Discussion

Con A-induced fulminant hepatitis is a well-known animal model for studying the pathophysiological mechanisms of acute liver failure, a devastating liver disease with significant mortality worldwide and without effective therapeutic approaches. Activated T cells, especially NKT cells, have been defined to play a critical role in promoting liver damage through producing cytokines, such as IFN-γ. However, the controlling mechanisms for NKT cells, especially for their cytokine production, have been elusive. In this report, we demonstrated for the first time, to our knowledge, that γδ T cells, especially Vγ4 γδ T cells, played a critical role in protective immune response against Con A-induced hepatitis through providing IL-17A, which, in turn, regulates NKT cell function, especially the production of IFN-γ.

One of the key findings in this study is establishing for the first time, to our knowledge, a protective role of γδ T cells in Con A-induced hepatitis. γδ T cells have many unique features and functions in comparison with conventional αβ T cells. It has been well documented that γδ T cells play an important role in many aspects of immune responses, including protective immunity against pathogens, tumor immune surveillance, and inflammatory diseases (9, 33). However, the role of γδ T cells in liver immune responses is unclear. Using either TCR δ−/− mice or transferring activated peripheral γδ T cells into TCR δ−/− mice, we collectively demonstrated that γδ T cells played a critical role in protective immune response against Con A treatment (Figs. 1, 5).

Furthermore, reconstitution of TCR δ−/− mice only with Vγ4, but not Vγ1, γδ T cells rendered these mice more protective against Con A-induced liver inflammation (Fig. 5). Our results added another example of divergent functions of different subsets of γδ T cells determined by their TCR usage. Interestingly, Vγ4 and Vγ1 γδ T cells are primarily in peripheral lymphoid organs (spleen and lymph nodes) (8); therefore, our study provided novel evidence that peripheral γδ T cells were involved in liver immune responses on Con A challenge. It is currently unclear whether these Vγ4 γδ T cells are newly recruited from periphery on Con A injection or prestored in the liver as “effector memory-like” αβ T cells. Given the fact that Vγ1 and Vγ4 γδ T cells have divergent functions in many other disease models, it remains to be determined whether Vγ4 γδ T cells play any similar roles in other LPS- or polyinosinic-polycytidylic acid-induced hepatitis models, and whether Vγ1 γδ T cells have any regulatory functions in these processes.

NKT cells are enriched in the liver and play an essential role in the pathogenesis of liver inflammation and damage, especially in the Con A-induced fulminant hepatitis model. To answer the question whether γδ T and NKT cells really “talked” to each other, we next provided evidence that the protective effect of γδ T cells indeed was through targeting NKT cells. We demonstrated that depletion of NKT cells completely abolished the severe liver damage in TCR δ−/− mice (Fig. 6A). Furthermore, the hallmark of proinflammatory cytokines IFN-γ (Fig. 6B, 6D), IL-4, and TNF-α (data not shown) expressed by NKT cells was significantly increased in TCR δ−/− mice in comparison with Wt mice on Con A injection, indicating a negative regulatory role of γδ T cells in the cytokine production by NKT cells. This effect was not NK1.1 dependent, because no significance was observed for cytokine expression level by NK1.1+CD3− NK cells between these two groups of mice (Fig. 6B). Both γδ T and NKT cells are innate-like lymphocytes, with an activated phenotype (34–36) and the ability to bridge the innate immunity and adaptive immune responses (23–25, 37, 38). Therefore, defining for the first time, to our knowledge, the interaction between these two cell types from this study, especially in the liver, which is a special organ with enriched immune cells, may have significant impact on understanding the immune pathology of other liver diseases. Further studies are granted to study the role of γδ T cells in other cell type-mediated liver damage and viral hepatitis.

What could be the critical mediator between γδ T and NKT cells? We hypothesized that it would be a cytokine. Indeed, our study firmly established that it was IL-17A deriving from Vγ4 γδ T cells that played a critical role in mediating the protective immune responses against Con A-induced liver damage. Our conclusion was supported by results obtained from multiple approaches. First, rIL-17 reconstituted the resistance against liver damage on Con A treatment in TCR δ−/− mice (Fig. 4A). Second, in comparison with those of Wt mice, liver tissues of TCR δ−/− mice showed a significantly decreased level of IL-17A (Fig. 3A), and Vγ4 γδ T cells expressed a high level of IL-17A (Fig. 5A, 5B). Third, only transferring Wt, but not IL-17A−/−, Vγ4 γδ T cells into TCR δ−/− mice offered protection against Con A-induced liver damage (Fig. 5G). Finally, consistent with findings described earlier, the regulation of the function of NKT cells by γδ T cells was IL-17A dependent (Fig. 6). Therefore, our results firmly established that Vγ4 γδ T cells provided the primary source of IL-17A and played a protective role in Con A-induced liver damage through targeting NKT cells. These results were also consistent with previous findings that Vγ4 γδ T cells were the main source of IL-17A on pathogen infection (27, 28). Interestingly, there were few, if any, CD4+ IL-17A+ cells, indicating a minor role of Th17 cells in this model. Given the critical role of NKT cells in Con A-induced hepatitis, and based on the fact that IL-17A–producing NKT cells express TCR αβ, but not NK1.1 (39), we also analyzed IL-17A production by αβ T cells. Similarly, a very low percentage of TCR αβ+ IL-17A+ T cells was detected in the Con A-treated liver (Fig. 3B), indicating that the critical role of NKT cells in this model was not through providing IL-17A. The protective role of IL-17A demonstrated in this article was also supported by a previous report that IL-17A protected against α-galactosylceramide-induced hepatitis in mice (40). However, in several other previous reports, there was no significant role for IL-17A in the Con A-induced hepatitis model using IL-17A−/− mice (41). The reason for such a discrepancy is unclear at the present time. We speculated that the main reason for such a discrepancy was due to the environment of the animal facility, which, in turn, resulted in the different intestine microbes, and consequentially led to the differential phenotypes. It has been reported that intestine microbes can influence IL-17A production by CD4+ T cells and experimental autoimmune encephalomyelitis (EAE) phenotype (42, 43). Further studies are needed to determine whether treatment of these mice with antibiotic will alter the phenotype.

Differing from those IFN-γ–producing Vγ4 γδ T cells as described in our previous studies (15–17), we speculated that these IL-17A–producing Vγ4 γδ T cells were “Ag -naive” γδ T cells. Further studies are needed to further define the functional differences between these two subtypes of Vγ4 γδ T cells and whether the interactions between these subtypes do exist.

Which function of NKT cells was regulated by γδ T cells? Because it has been well established that IFN-γ is a critical cytokine in the pathogenesis of Con A-induced hepatitis (3, 5), and it was reported that IL-17A can downregulate T-bet and IFN-γ in CD4+ T cells (44), we hypothesized that Vγ4-derived IL-17A might downregulate IFN-γ production by NKT cells. Indeed, in the absence of γδ T cells, NKT cells expressed a significantly higher level of IFN-γ (Fig. 6B, 6D), and transferring Wt, but not IL-17A−/−, Vγ4 γδ T cells into TCR δ−/− mice completely decreased IFN-γ expression from NKT cells (Fig. 6C, 6D). We speculated that Vγ4-derived IL-17A might act on NKT cells to alter IFN-γ–producing programs. Further studies are needed to give us a better clue about the detail of molecular mechanisms.

In summary, our study has demonstrated a critical role of γδ T cells, especially Vγ4 γδ T cells, in protection against Con A-induced hepatitis. This protective effect was mediated by Vγ4 γδ T cell-derived IL-17A, which, in turn, targeted NKT cells and negatively regulated IFN-γ production by NKT cells. Thus, for the first time, to our knowledge, our results define a critical role of γδ T cells in Con A-induced hepatitis and show that transferring γδ T cells may provide a novel therapeutic approach for this devastating liver disease.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We are grateful to Dr. Mark Bartlam and Marie-Louise Hjaeresen for editing the manuscript. We also greatly appreciate technique support by Xinglong Zhou and Qiang Zhao from the core facility of College of Life Sciences, Nankai University.

Footnotes

  • ↵1 N.Z., J.H., and Y.N. are cofirst authors.

  • This work was supported by the National Outstanding Young Scientist Award of the National Natural Science Foundation of China (Grant 30725015 to Z. Yin; Grant 31000400 to Z.W.), the National Basic Research Program of China (Grant 2007CB914801 to Z. Yin; Grant 2010CB945003 to J. Han), and the Tianjin Municipal Science and Technology Commission (Grant 07ZCKFSH03600 to Z. Yin; Grant 09ZCKFSH08200 to Z.H.).

  • Abbreviations used in this article:

    ALT
    alanine aminotransferase
    ASGM1
    anti-asialo GM1
    MNC
    mononuclear cell
    rm
    recombinant mouse
    RPLP0
    ribosomal protein large P0
    Wt
    wild-type.

  • Received May 5, 2011.
  • Accepted September 5, 2011.

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